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Phage Titering

Goal: want to know approximately how many phage are within a given solution volume (ex: # phage/mL)

Phage serial dilutions

  • Have your phage lysate (~1e10 or 1e11 phage/mL) in fridge already
  • Get 5 test tubes out
  • Label tubes: (1) 1e2, (2) 1e4, (3) 1e6; (4) 1e7, (5) 1e8
  • fill tubes 1-3 with 9.9mL LB solution
  • fill tubes 4-5 with 9 mL LB solution
  • add 100 microL phage lysate to tube 1 and mix well
  • add 100 microL tube 1 into tube 2 and mix well
  • add 100 microL tube 2 into tube 3 and mix well
  • add 1 mL tube 3 into tube 4 and mix well
  • add 1 mL tube 4 into tube 5 and mix well

  • NOTE: We only are going to use dilutions 4 & 5. Dilutions 1-3 were only created to get to 4 & 5.

Soft agar prep

  • get 8 small test tubes with metal caps and put into heating block @55C
  • put 2.5 mL of liquid soft agar (found in 55C incubator) into each tube and continue heating in block

Soft agar container prep

  • get out 8 - 1.7 mL eppendorf tubes and label 1-4 with 1e7; 5-8 with 10e8... 1-2 & 5-6 with 50 microL and 3-4 & 6-7 with 100 microL
  • get out 8 petri dishes and label them the same as each eppendorf tube marking along a 1 or 2 denoting duplicate number

Phage infection of bacteria in soft agar

Ultimately we want to create a plate full of bacteria and mix in a known volume of diluted phage solution to let the phage infect the bacteria and create a discrete number of plaques that can be visualize and counted to determine the actual number of phage that was in a particular volume of phage solution. This is a

  • put 250 microL overnight bacteria stock into each eppendorf tube
  • put 50 or 100 microL phage dilutions into each eppendorf tube for the appropriately labeled tube
  • put entire mixed eppendorf tube contents into soft agar tube
  • pour soft agar tube immediately into petri dish
  • place petri dishes in 37C room and flip dishes when agar hardens

Counting phage plaques

After overnight growth, visual plaques should be apparent and can be hand counted. Thus, we can calculate the true value of phage concentration by: (# plaques)/(X microL solution) <-- Depends on volume of phage solution added (50 or 100)